Intestinal intraepithelial lymphocytes (IELs) form one of the key branches of the mucosal immune system, potentially providing a first line of immune defense against pathogens due to their location at the critical interface between the intestinal lumen and the core of the body. Although in recent years some of the mechanisms controlling the development of different IEL populations have been elucidated, the role played by IELs during gastrointestinal infection remains elusive. A few main obstacles hamper efforts to address these deficits: lack of genetic models that would allow specific control of IEL development and function, poor IEL survival ex vivo and difficult access due to their location within the epithelial compartment. This proposal addresses these main impediments using a combination of novel tools and approaches to dissect the function of these cells in vivo. First, to address IEL dynamics in vivo we will use multi-photon intra-vital as well as tissue clearing associated with light sheet microscopy. Using these techniques we have been able to track IEL dynamics in multiple villi simultaneously in nave animals and during enteric infections. We observed that TCR??+ IELs are highly mobile cells that constantly survey the intestinal epithelium, rapidly responding to pathogenic bacterial infection by changing their motility and pattern of shuffling between intestinal epithelial cells (IECs), as well as their location within the villi. These changes were linked to gene expression changes and changes in energy utilization pathways. We hypothesize that a coordinated IEC-IEL response to luminal perturbations results in modification of IEL behavior and metabolism, ultimately leading to optimal resistance to pathogen invasion. Mostly focusing on the main IEL population, TCR??+ cells, the studies in this proposal will define they respond to intestinal microbes utilizing gnotobiotic mice as well as models of gastrointestinal infections. We will characterize the relative contribution of IEL cell dynamics and metabolic changes in response to enteric pathogens to their protective function. We will also study how sensing of microbes by IECs influences IEL responses against infections. Ultimately, using gene-targeting strategies and molecular biology techniques, we will investigate the role of the transcription factor T-bet in the modulation of activity of IEL subsets and the underlying molecular mechanisms. By combining gene-reporter, multiple cell-specific and temporally controlled gene targeting and in vivo imaging strategies with various model of microbial stimulation, we expect to identify specific surveillance and protective characteristics for IELs. This proposal offers completely novel approaches to dissect the function T cells that constantly scan the intestinal surface. Thus, the knowledge gained from this study will expand our understanding of adaptive immunity at the gut epithelial barrier, contributing valuable information regarding host-microbial interactions. Defining key IEL functions during intestinal defense is an important step for understanding the initial processes involved in mucosal immunity and may lead to better strategies for therapeutic intervention in gastrointestinal infections.